KR100396841B1 - Composition of season for Danmuji with anticancerous function and Danmuji thereof - Google Patents

Abstract

The present invention relates to a seasoning composition for anticancer functional radish and seasoned anticancer functional radish using the same as gardenia (gardenia yellow) and safflower (safflower yellow) with salt, salt-baked salt and KCl alternative salt, stevioside as sugar The anticancer functional radish of the present invention seasoned and aged using sodium alginate with salt adsorption function as a stevioside) and an active substance inhibits solid cancer growth, enhances the immune function of the spleen, and removes toxic substances from the liver. -S-transferase (glutathione-S-transferase) increases the activity of the excellent anti-cancer effect is excellent.

Description

Seasoning composition for anticancer functional radish and anticancer functional radish seasoned using the same {Composition of season for Danmuji with anticancerous function and Danmuji

The present invention relates to a seasoning composition for anticancer functional radish and anticancer functional radish seasoned using the same. More specifically, the present invention relates to natural pigments, roasted salt, KCl substitute salt, stevioside and sodium alginate during production of radish. (Sodium alginate) relates to the seasoning composition for radish enhanced anti-cancer effect using the anti-cancer and anti-cancer functional radish seasoned using the same.

Pickled radish is rarely immersed in homes, and is almost commercially immersed in factories. The production method of pickled radish is mainly distributed by using salt immersion method, simply salted radish, and then desalted to maintain proper salinity after a certain period of time (2-3 months). There is a problem that the color changes little by little regardless of product deterioration by enzymatic decomposition and oxidation during distribution.

On the other hand, since the taste of pickled radish is mainly felt as salty and sweet, the choice of raw materials for these two flavors is very important. However, commercially available pickled radish uses the salt of the sun salt and the salt of sodium saccharin (sodium saccharin). Natural salts are heavy metals due to sea pollution these days, and sodium saccharin is not preferable as a food additive because it can cause cancer when overdose.

In order to solve the above problems, the present inventors refined the natural pigments of gardenia and safflower yellow so that the radish does not degrade the product even if browning occurs to some extent, and salt is purified at high temperature as a substitute for sun salt. Roasted salt and KCl alternative salts free of impurities are used, sugar, stevioside, a natural sweetener, and sodium alginate, which are known to have salt adsorption, are used as a substitute for sodium saccharin. The present invention was completed by confirming that the prepared radish had anti-cancer effects.

Accordingly, an object of the present invention is to provide a seasoning composition for anticancer functional radish prepared using natural pigment, baked salt, KCl substitute salt, stevioside, sodium alginate, and anticancer functional radish using the same. .

The object of the present invention is to prepare the anticancer functional radish of the present invention using natural pigments, roasted salt and KCl mixed salt, stevioside and sodium alginate and to produce commercially available radish by different materials in the same manner. The anticancer functional radish and commercial radish of the present invention were then administered to mice transplanted with tumor cells, and the weight of solid cancer, the activity of natural killer cells, and the activity of glutathione-S-transferase. Investigate each of the anticancer functional radish of the present invention has anticancer properties and examine the weight change of the mouse organs to investigate the effect of the radish extract of radish on other organs besides the effect of enhancing anticancer activity and the anticancer functional radish of the present invention Distribution of Glycogen and Immunohistologic Responses in Mice Treated with Methanol Extract It was achieved by irradiation.

Hereinafter, the configuration and operation of the present invention.

1 is a flow chart showing the manufacturing process of the anticancer functional radish of the present invention.

Figure 2 is a graph showing the effect of the anticancer functional radish and commercial radishes of the present invention on natural killer cell activity of mouse spleen lymphocytes.

Figure 3 is a graph showing the effect of the anticancer functional radish and commercial radish of the present invention on glutathione-S-transferase activity in mouse liver.

4A to 4D show methanol extracts of group (b), commercial radish (c), and anticancer functional radish (d) of the present invention, in which only PBS was administered to mice transplanted with normal liver tissue (a) and tumor cells sarcoma-180, respectively. The result of confirming the glycogen distribution in the liver tissue of the group administered by hematoxylin eosin staining.

5A to 5D show methanol extracts of group (b), commercial radish (c), and anticancer functional radish (d) of the present invention, which were administered with PBS only to mice transplanted with normal liver tissue (a) and tumor cells sarcoma-180, respectively. Glycogen distribution in the hepatic tissue of the group administered is a photographic view of the result of the pyiodic acid Schiff (PAS) reaction.

6a to 6d are methanol extracts of the group (b), commercially available radish (c), and anticancer functional radish (d) of the present invention, which were administered with PBS only to mice transplanted with normal liver tissue (a) and tumor cells sarcoma-180, respectively. It is a photograph of the response result shown to antibody Bax among tumor-related proteins with respect to liver tissue of administration group.

7A to 7D are methanol extracts of the group (b), commercial radish (c), and anticancer functional radish (d) of the present invention, which were administered with only PBS to mice transplanted with normal liver tissue (a) and tumor cells sarcoma-180, respectively. The photograph shows the result of the reaction shown in antibody Rb (sc-050) among tumor-associated proteins to the liver tissues of the administered group.

8a to 8d are methanol extracts of the group (b), commercial radish (c), and anticancer functional radish (d) of the present invention, which were administered with PBS only to mice transplanted with normal liver tissue (a) and tumor cells sarcoma-180, respectively. The photograph shows the result of the reaction shown in the antibody c-fos (sc-253) among the tumor-related proteins to the liver tissue of the group to which the administration was performed.

The present invention prepares the anticancer functional radish of the present invention using natural pigments, roasted salt and KCl mixed salt, stevioside and sodium alginate, and the same method as the anticancer functional radish of the present invention using sun salt and saccharin without using a pigment. Preparing commercial radish; Lyophilizing commercially available radish and the anticancer functional radish of the present invention, followed by pulverization to obtain methanol extract by adding 10-fold methanol; Implanting tumor cells into the mouse and administering a commercial radish extract and an anticancer functional radish extract sample of the present invention, and then measuring the tumor weight to investigate the effect of inhibiting solid cancer growth of the anticancer functional radish of the present invention; Investigating the effect of the sample on other organs by measuring the weight change of each organ when PBS was administered to a mouse transplanted with tumor cells and when the commercial radish extract and the anticancer functional radish extract sample of the present invention were administered; Investigating the activity of natural killer (NK) cells in the spleen of mice transplanted with tumor cells to investigate the effect of increasing the immune activity of the anticancer functional radish of the present invention; Measuring the enzyme glutathione-S-transferase activity involved in detoxification by transferring and excreting toxic substances and peroxides in the liver of mice transplanted with tumor cells; Investigating glycogen distribution in liver tissue of a mouse transplanted with tumor cells to examine the degree of hepatocellular damage of the anticancer functional radish of the present invention; Investigating immunohistochemical responses to tumor-related proteins in liver tissues of mice transplanted with tumor cells. The natural pigment used in the present invention is to prevent the quality of the product from deteriorating even if the brown radish is browned to some extent. gardenia yellow) and safflower yellow were mixed at a ratio of 3: 7 to 7: 3, and 0.2 to 0.6% by weight of the total weight of the seasoning composition for anticancer functional radish was used.

Salt used in the present invention is a high-temperature refined as a substitute for sun salt and baked salt and KCl substitute salt (containing KCl and NaCl as 3: 7) in a ratio of 7: 3 to 3: 7, and the anti-cancer functional radish seasoning 1-3 weight percent was used relative to the total weight of the composition.

The sugar used in the present invention used stevioside, a natural sweetener, as a substitute for saccharin sodium, and 0.1 to 0.4% by weight based on the total weight of the seasoning composition for anticancer functional radish.

The active material used in the present invention used sodium alginate known to have a salt adsorption function, 0.3 to 0.7% by weight based on the total weight of the anti-cancer functional radish seasoning composition and the total weight of the seasoning composition for anticancer functional radish The anticancer functional radish of the present invention was prevented from browning of the radish by using natural pigments that were beneficial in the seasoning process after the desalting process was completed. Taste was added, and it also played an important role in anticancer enhancement.

Hereinafter, the specific method of the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited only to these Examples.

Example 1: Anticancer functional radish and commercial radish preparation of the present invention

Autumn radish was washed clean and immersed in mixed salt brine mixed with 15% of roasted salt and KCl salt in a ratio of 3: 7, then completely desalted, seasoned with pigments, active substances, sugars and salts, and aged. At this time, the seasoning composition is shown in Table 1 as a pigment used in the gardening yellow and safflower yellow (7: 3) ratio, salt is KCl alternative salt (KCl: NaCl = 3: 7) and baked salt 3: The ratio was 7 and stevioside (stevioside 100s) was used as sugar and sodium alginate (0.5% sodium alginate) as an active substance. After seasoning was soaked and aged in the refrigerator for 4 days (Fig. 1). Commercial radish also produced in the same manner as the anticancer functional radish of the present invention, but the materials used are also shown in Table 1.

Material mixing ratio of anticancer functional radish and commercial radish of the present invention Commercial pickled radish Anticancer functional radish Desalted radish 1 kg 1 kg water 1 kg 1 kg Citric acid 0.6kg 0.6g Acetic acid crystals 2 g 2 g Party Saccharin 0.6g Stevioside 1.8g Glycine 0.26 g 0.26 g Vitamin c 0.166 g 0.166 g Malic acid 0.266 g 0.266 g Salt ¹ 20 g Baked salt 14g: KCl + NaCl (KCl: 1.8g, NaCl: 4.2g) Phosphate (STPP) 0.2 g 0.2 g Natural colors ² - Yellow tea (1.4-4.2g) + yellow safflower (0.6-1.8g) Active substance - Alginate (0.5%) 5g 1 Salt: Baked salt: Alternative salt = 7:32 Natural color: Gardenia yellow: Safflower yellow = 7: 3 (gardenia yellow) (safflower yellow)

Example 2: Preparation of Methanol Extract of Seasoned Radish

In Example 1, four days of immersion and maturation of the dried radish were aged by lyophilization and pulverization, and then methanol was added 10 times the weight of the sample, and the sample for 3 to 4 hours in a 60 ~ 70 ℃ water bath. This process was repeated three times for extraction. The methanol extract obtained here was concentrated in a rotary vacuum concentrator to completely remove the methanol. The sample used in the experiment was dissolved in 10% solution in DMSO (dimethyl sulfoxide), frozen in a freezer, thawed if necessary, and diluted to an appropriate concentration.

Example 3 Solid Cancer Growth Inhibition Effect of Anticancer Functional Radish of the Present Invention

In this example, in order to see the anti-tumor effect of the anticancer functional radish obtained from Example 2 and the methanol extract obtained from commercial radish obtained in Example 2, sarcoma-180 tumor cells were implanted 20 subcutaneously under the left groin of Balb / c mice. Samples were administered for one day and 32 days later, mice were dissected to isolate tumors and weighed. That is, based on the viability test (viblility test) in vitro , both the functional and commercial radish was determined at a concentration of 0.25mg / mL and 0.25mg / kg of radish methanol extract was administered as a sample. As a result, as shown in Table 2, the control group implanted with only tumor cells (S-180 + PBS, administered with the same amount of PBS instead of the sample) had a tumor weight of 6.08g while the group was injected with functional mussels (S-180 + FD) decreased to 4.38 g, showing 27.96% of tumor suppression effect. The group injected with commercially available radish (S-180 + CD) was 5.93 g, which was not significantly different from the control group. Therefore, the functional radish was found to have a solid growth inhibitory effect.

Tumor weight measurement after administration of radish methanol extract to Balb / c mice transplanted with sarcoma-180 cells Sample Dose (mg / kg) Tumor Weight (g) Inhibition Ratio (%) S-180 + PBS 0.25 6.08 ± 0.04 - S-180 + CD ¹ 0.25 5.93 ± 0.84 2.47 S-180 + FD ² 0.25 4.38 ± 1.22 27.96 [Note] 1: Commercial radish radish 2: Anticancer functional radish radish

Example 4 Weight Changes of Organs of Rats Fed Methanol Extract by Diet

In order to measure the action of the anticancer functional radish methanol extract and commercial radish methanol extract of the present invention in other organs besides the tumor, the control was only free diet, and the tumor cell control group (S-180 + PBS) was free diet. PBS was administered by injection, and the treated group was given a free diet and injected the sample for 20 days after transplanting tumor cells (Sarcoma-180 cell) with the same extract of radish as the tumor cell control group. And the weight of each organ was measured. As a result of the experiment, as shown in Table 3, the weight ratio of liver, which is responsible for the detoxification of various harmful substances generated during metabolic processes, was the control group (S-180 + PBS) transplanted with tumor cells only and the group injected with commercially available anchovy (S- 180 + CD) was slightly higher than the group injected with functional monoclonal (S-180 + FD) and the spleen was significantly lower than the control group implanted with only tumor cells. There was no big difference.

Weight of each organ of mice fed with methanol extract of anticancer functional radish and commercial radish methanol extract of the present invention as a diet Sample Weight (g) Spleen / Weight (%) Liver / Weight (%) Heart / Weight (%) Height / weight (%) Control 28.95 ± 1.9 0.33 ± 0.02 5.74 ± 0.6 0.50 ± 0.15 1.85 ± 0.05 S-180 + PBS 29.5 ± 1.3 1.55 ± 0.12 7.72 ± 0.9 0.34 ± 0.01 1.58 ± 0.04 S-180 + CD ¹ 29.9 ± 1.4 1.47 ± 0.09 7.57 ± 0.7 0.34 ± 0.01 1.70 ± 0.18 S-180 + FD ² 28.6 ± 2.1 1.40 ± 0.01 6.68 ± 0.4 0.35 ± 0.03 1.66 ± 0.07 [Note] 1: commercially available radish 2: anti-cancer functional radish

Example 5 Increasing Immune Activity of Anticancer Functional Radishes and Commercial Radishes of the Present Invention

In this embodiment, tumor cells were transplanted in the same manner as in Example 4, and after administration of the anticancer functional radish extract and commercial radish extract sample of the present invention for 20 days, spleens of each mouse were sterilely extracted and natural killing of spleen was performed. killer (NK) cell activity was examined. In order to analyze the activity of natural killer cells, first, effector cells (NK cells) were prepared. The spleens of the mice were aseptically extracted in a clean bench, washed three times with 5 mL of RPMI 1640 medium containing penicillin (100 U / mL) and streptomycin (100 μg / mL) and then finely ground. The cell suspension was filtered through a 70 μm nylon mesh, centrifuged to collect lymphocytes, and then suspended in the same medium. Lymphocytes were isolated by centrifugation (500 × g, 30 min, 18 ° C.) using histopacque-1077 (histopaque-1077). The prepared cells were resuspended in RPMI 1640 medium containing 10% FCS, 2 mM L-glutamine, penicillin (100 U / mL), streptomycin (100 μg / mL). This was incubated for 1 to 2 hours at 37 ° C. in a CO ₂ incubator to allow cells to adhere to the culture flask (6 cm Petri dish). Non-adhesive natural killer cells were collected by centrifugation, resuspended in culture medium, and the appropriate number of cells was counted. Subsequently, cell destruction activity of natural killer cells was analyzed by MTT method. Yac-1 mouse lymphoma cells were used as target cells. Yac-1 cells are the most convenient target cell line for measuring NK cell activity. Yac-1 cells were diluted to a density of 5 × 10 ⁴ cells / mL in RPMI 1640 medium containing 10% FCS, 2 mM L-glutamine penicillin (100 U / mL) and streptomycin (100 μg / mL). 50 μl was added to the wells. And NK cells were added to the 96 well plate 50μL of effector cells and Yac-1 cells for the analysis of NK cell activity of 1 × 10 ⁷ cells / mL. After incubating for 3 days at 37 ° C. in a CO ₂ incubator, 10 μl of MTT (5 mg / mL) solution was added to each well and incubated at 37 ° C. for 4 hours. 25 μl of SDS (10% in 0.02N HCl) was added thereto, followed by color development at room temperature for 30 minutes, and OD was measured at 540 nm. Percent cytotoxicity was calculated by the following formula.

As a result, as shown in Figure 2, the anti-cancer functional group (S-180 + FD) showed the highest inhibitory effect of 58.39%, the control group was 50.37%, only the tumor cells were transplanted 32.01%, tumor cells were transplanted The group receiving the commercial radish methanol extract showed 32.41%. In the above results, the group administered with commercial radish showed low NK cell immune activity, similar to the group transplanted with only tumor cells.

Example 6: Glutathione-S-transferase Activity Changes in Mouse Liver by Anticancer Functional Radish and Commercial Radish of the Present Invention

Phase II of the metabolic enzyme system in the liver removes foreign substances by including endogenous substances or toxic substances administered externally or by converting them into water-soluble substances to be discharged to the body. Glutathione-S-transferase (glutathione-S- Transferase is an enzyme that is involved in detoxification by transferring and excreting toxic substances and peroxides in the body using reduced glutathione.

In the present Example, glutathione-S-transferase activity in the rats fed the sample and the diet was measured as in Example 5. First, mice were killed to measure glutathione-S-transferase (GST) activity, and then liver was perfused with physiological saline of 4 ° C. or lower to remove blood remaining in the liver, followed by extraction of the liver. 0.1 M potassium phosphate buffer (pH 7.4) was added per 1 g of liver tissue, and the mixture was ground with a glass teflon omogenizer under ice cooling. This grinding solution was used as a homogenate fraction, which was centrifuged at 13,000 rpm for 10 minutes to remove the nuclei and unbranched cell sections, and the supernatant obtained by ultracentrifugation for 1 hour at 105,000 × g was used as the cytosol fraction. It was. According to the method of Habig et al., 75 μl of 0.04 M reduced glutathione in 0.1 M potassium phosphate buffer (pH 6.5) was added, 0.1 mL of enzyme solution was added, 0.5 mL of 20% trichloroacetic acid was added to the blank, and the sample was inactivated at 25 ° C. After reacting for 5 minutes, add 25 µl of 0.12M 1-chloro-2,4-dinitrobenzene to the blank and each sample, and react for 2 minutes at 25 ° C. Then, add 20% trichloroacetic acid to the sample to complete the reaction. The supernatant obtained from the separation was measured for absorbance at 340 nm, and then the activity was calculated using the molar extinction coefficient of 1-chloro-2,4-dinitrobenzene at 9.6 mM ¹ cm ^-1 . Units of enzymatic activity were expressed as nmole number of 2,4-dinitrobenzene-glutathione produced by mg protein for 1 minute. As shown in FIG. 3, glutathione-S-transferase activity was 392.2 nmol / mg protein / min in the control group without transplanting tumor cells and 281.4 nmol in the control group (S-180 + PBS) without transplantation of tumor cells. dropped to / mg protein / min. In the group injected with commercially available radish (S-180 + CD), the level was lower than the control group implanted with tumor cells at 277.5 nmol / mg protein / min. The activity of Glutathione-S-transferase was significantly higher than that of the control group transplanted with 510.4 nmol / mg protein / min.

Example 7 Investigation of Glycogen Distribution in Mouse Liver Tissues by Anticancer Functional Radish and Commercial Radish

Intrahepatic Tissue Changes in Mice Group / HC CS HD FC KC FN Normal + 0- + +-++ + 0- + S-180 + PBS +++ ++ +++ +++ ++ S-180 + CD +++ ++ +++ +++ ++ S-180 + FD +++ ++ ++ ++ + Note: HC: histologic change, CS: boil formation, HD: hydrophilic alteration, FC: lipid change, KC: Kupffer cell response, FN: lesion regression 0: no change or negligible +: appears weak ++: moderate Shown as a level +++: Shows bad ++++: Shows very bad

Glycogen Distribution in Mouse Liver group PZHL IZHL CZHL Normal ++++ ++-+++ ++-+++ S-180 + PBS +++ ++ ++-+++ S-180 + CD ++-+++ ++-+++ ++-+++ S-180 + FD +++ ++-+++ ++-+++ Note: PZHL: Peripheral section of hepatic lobe, IZHL: Intermediate section of mesenchymal lobe, CZHL: Central section of mesenchymal lobe, 0: No change or negligible +: Appear weak +++: Appear moderate ++++: Very bad

Example 8: Immunohistologic Responses of Mouse Liver Tissues by Anticancer Functional Muds and Commercial Muds of the Invention

In this example, to see the immunohistochemical response of the tumor-associated protein to the liver tissue of the rat fed the sample and diet as in Example 5, deparaffinizing 10 mM for immunohistochemical observation of the protein associated with each tumor Treated at 95 ° C. for 5 minutes in sodium citrate buffer (pH 6.0) and again placed in the same buffer at room temperature for 20 minutes. This was treated with 3% methanol hydroperoxide at room temperature for 30 minutes, washed three times with PBS (0.01M, pH 7.5) three times for 5 minutes, followed by goat normal serum (Vector Lab., PK-6101). Treatment was at room temperature for 30 minutes. Remove it and replace Bax (sc-493), Bcl-2 (sc-783), Rb (sc-050), c-fos (sc-253) and c-jun (sc-1694) from Santa Cruz Biotechnology Inc. Diluted to 500: 1 and reacted for 4 hours in 4 ℃ wet. Washed with PBS and reacted with biotinylated anti-rabbit lg G (Vector Lab., PK-6101) for 30 minutes at room temperature, washed with PBS and ABC kit (avidin-biotin-peroxidae complex, vector Lab., PK-6101) Was reacted at room temperature for 60 minutes. After washing with PBS again, it was developed for 5 minutes at room temperature with a DAB substract kit (vector Lab., SK-4100). After washing with distilled water and control dyeing with Mayer's hematoxylin, the reaction degree was determined under an optical microscope. As a result, immunohistochemical reactions of tumor-associated proteins against hepatocytes showed only Bax, Rb, and c-fos among six antibodies, and showed stronger responses in the nucleus than cytoplasm, and also in inflammatory cells between some hepatocytes. Indicated. As shown in FIGS. 6a to 6d, Bax showed a reaction only in the nucleus, and S-180 + PBS and S-180 + CD were decreased compared with the normal group, and the decrease was small in the functional group of S-180 + FD. As shown in Figure 7a to 7d Rb increased in the case of S-180 + PBS and S-180 + CD in the nucleus and cytoplasm compared to the normal group, especially the change in the cytoplasm of the peripheral venous hepatocytes, but S functional group In the case of -180 + FD, cytoplasmic changes were small. c-fos is increased in S-180 + PBS compared to the nucleus and cytoplasm of the normal group as shown in Figures 8a to 8d, but also similar to the control group in the commercial radish group S-180 + CD and S-180 + FD It was. This is summarized in Table 6.

Immunohistochemical Responses of Tumor-associated Proteins in Rat Liver group Region / Antibody Bax Bcl-2 Rb c-jun c-fos Normal Nuclear cytoplasm ++++ 0 00 +++ 0- + 00 +++ 0- + S-180 + PBS Nuclear cytoplasm ++ 0 00 ++++++ 00 ++++++ S-180 + CD Nuclear cytoplasm ++-+++ 0 00 +++++ 00 ++++ S-180 + FD Nuclear cytoplasm +++ 0 00 ++++ 00 ++++-++ Note: 0: No change or negligible +: Appears weak +++: Appears moderate +++: Appears severe +++: Appears very bad

As described above, as described in the above examples, gardenia (gardenia yellow) and safflower (safflower yellow), salt-to-salt salt and KCl substitute salt, stevioside as sugar, and a salt adsorbing function as an active substance as described in the above embodiment The anticancer functional radish of the present invention seasoned and aged using sodium alginate increases the activity of glutathione-S-transferase which inhibits solid cancer growth, enhances the spleen's immune function, and removes toxic substances from the liver. It is a very useful invention in the food industry because it has an excellent effect of showing excellent anticancer properties.

Claims (4)

delete 0.2 to 0.6% by weight of mixed pigments of gardenia yellow and safflower yellow in a ratio of 7: 3, and 1 to 3 weight of salt mixed with roasted salt and KCl alternative salt in a ratio of 7: 3 to 3: 7 %, Stevioside (stevioside) 0.1 to 0.4% by weight, sodium alginate (sodium alginate) 0.3 to 0.7% by weight, water 95.3 to 98.4% by weight seasoning composition for anti-cancer functional radish. An anticancer functional pickled radish characterized by seasoning and ripening radish desalted by the seasoning composition for anticancer functional radish described in claim 2. The washed radish was immersed in 15% brine mixed with roasted salt and KCl in the range of 3: 7 to 7: 3, and then completely desalted, seasoned with the anticancer functional radish seasoning composition as described in claim 2, and soaked in a refrigerator for 4 days. Anticancer functional pickled radish prepared by aging.